Journal Pre-proofs

Review

Advances in developing metabolically engineered microbial platforms to pro‐

duce fourth-generation biofuels and high-value biochemicals

Muhammad Aamer Mehmood, Ayesha Shahid, Sana Malik, Ning Wang,

Muhammad Rizwan Javed, Muhammad Nabeel Haider, Pradeep Verma,
Muhammad Umer Farooq Ashraf, Nida Habib, Achmad Syafiuddin, Raj
Boopathy

PII: S0960-8524(21)00850-6
DOI: https://doi.org/10.1016/j.biortech.2021.125510
Reference: BITE 125510

To appear in: Bioresource Technology

Received Date: 28 May 2021

Revised Date: 1 July 2021
Accepted Date: 2 July 2021

Please cite this article as: Aamer Mehmood, M., Shahid, A., Malik, S., Wang, N., Rizwan Javed, M., Nabeel
Haider, M., Verma, P., Umer Farooq Ashraf, M., Habib, N., Syafiuddin, A., Boopathy, R., Advances in
developing metabolically engineered microbial platforms to produce fourth-generation biofuels and high-value
biochemicals, Bioresource Technology (2021), doi: https://doi.org/10.1016/j.biortech.2021.125510

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1 Advances in developing metabolically engineered microbial platforms to produce

2 fourth-generation biofuels and high-value biochemicals

4 Muhammad Aamer Mehmood a, b, Ayesha Shahid b, Sana Malik b, Ning Wang a, Muhammad

5 Rizwan Javed b, Muhammad Nabeel Haider b, Pradeep Verma c, Muhammad Umer Farooq

6 Ashraf b, Nida Habib b, Achmad Syafiuddin d, Raj Boopathy e*

8 a School of Bioengineering, Sichuan University of Science and Engineering, Zigong, China

9 b Bioenergy Research Centre, Department of Bioinformatics & Biotechnology, Government

10 College University Faisalabad, Faisalabad, Pakistan

11 c Department of Microbiology, Central University of Rajasthan, Bandarsindri, Kishangarh,

12 Ajmer-305801, Rajasthan, India

13 d Department of Public Health, Universitas Nahdlatul Ulama Surabaya, 60237 Surabaya, East

14 Java, Indonesia

15 e Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310, USA

16

17 *Corresponding author: ramaraj.boopathy@nicholls.edu

18

19 Running Title: Microbial platforms to produce fourth-generation biofuels

20

21

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23

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25

1
26 Abstract

27 Producing bio-based chemicals is imperative to establish an eco-friendly circular

28 bioeconomy. However, the compromised titer of these biochemicals hampers their

29 commercial implementation. Advances in genetic engineering tools have enabled researchers

30 to develop robust strains producing desired titers of the next-generation biofuels and

31 biochemicals. The native and non-native pathways have been extensively engineered in

32 various host strains via pathway reconstruction and metabolic flux redirection of lipid

33 metabolism and central carbon metabolism to produce myriad biomolecules including

34 alcohols, isoprenoids, hydrocarbons, fatty-acids, and their derivatives. This review has briefly

35 covered the research efforts made during the previous decade to produce advanced biofuels

36 and biochemicals through engineered microbial platforms along with the engineering

37 approaches employed. The efficiency of the various techniques along with their shortcomings

38 is also covered to provide a comprehensive overview of the progress and future directions to

39 achieve higher titer of fourth-generation biofuels and biochemicals while keeping

40 environmental sustainability intact.

41

42 Keywords: advanced biofuels; biomolecules; heterologous synthesis; improved titers;

43 genetic engineering.

44

45 1. Introduction

46 The previous decade has seen an upsurging trend in biofuel and high-value

47 biochemical production to reduce dependence on non-renewable and synthetic sources, to

48 lower the damaging environmental impacts, and to promote biorefinery. It is predicted that

49 the biochemical market will escalate from 2% to 20% by the year 2025 due to the growing

50 demands for biobased products which has led to the R&D efforts to focus on commercially-

2
51 oriented research trends (Cordova et al., 2020). In recent years, renewable biofuels have seen

52 significant potential amid the environmental and financial crisis of fossil fuels. Petroleum-

53 replica molecules like hydrocarbons, isoprenoids, fatty-acid derived molecules, and higher

54 alcohols have an advantage over their conventional counterparts due to engine compatibility,

55 higher density, and compatibility with existing storage and transport infrastructure. Whereas

56 biologically active high-value compounds including isoprenoids and fatty acid-derived

57 biomolecules are of biotechnological, pharmacological, agricultural, environmental, and

58 industrial importance (Adegboye et al., 2021).

59 Depending upon the biofuel/chemical molecule, it either requires sugary/starchy

60 substrate or lipid-rich biomass for their subsequent conversion into higher alcohols,

61 bioethanol, butanol, isoprenoids, hydrocarbons, and fatty-acid derived molecules (Zada et al.,

62 2018). First-generation feedstocks include food crops such as sugarcane, corn, beetroot,

63 wheat, barley, etc. however it sparked the debate of food shortage. Second-generation

64 feedstocks focused on raw and waste materials to address the issues of 1st generation

65 feedstock but they also required costly and time-consuming pretreatments for their

66 decomposition into simpler units for their easier subsequent conversion to products. The third

67 generation considered photosynthetic microbes such as microalgae and cyanobacteria as

68 feedstock to produce both higher alcohols and lipid-based products (Hammer et al., 2020).

69 Microalgae gained popularity over lignocellulosic biomass due to their biochemical

70 composition which consists of carbohydrate, lipid, and protein content. Carbohydrate content

71 is feasible to produce higher alcohols and bioethanol while, lipid fraction is utilized to

72 produce biodiesel, isoprenoids, and other lipid-based compounds. However, commercial

73 implementation of 3rd generation feedstock is limited due to constraints of the cultivation,

74 harvesting, and downstream processing (Tarafdar et al., 2021). The shortcomings of the

75 previous generations were believed to be addressed by genetically modifying microbes

3
76 (termed as 4th generation) to improve the process efficiency and the product yield. The

77 previous decade has seen an upsurge in biofuel and high-value biochemical production by

78 employing microbial cell factories (Phulara et al., 2018). Microbial cell factories are a

79 reliable sustainable source with the potential to reduce environmental footprint, therefore,

80 these have been extensively employed to achieve the commercial production of biopolymers

81 like polyhydroxyalkanoates (Sindhu et al., 2021) and various industrially important enzymes

82 (Tarafdar et al., 2021). These cell factories provide the opportunity to move from a fossils-

83 oriented economy to a bioeconomy (Adegboye et al., 2021). However, their production

84 remained unable to meet the commercial demands due to low titer, which is possible because

85 of slow growth rate, carbon source utilization inability, cofactor imbalance, inefficient

86 genetic regulation, competitive pathways, evolutionary loss of metabolic abilities, and

87 cellular toxicity. Genetic engineering approaches to manipulate microbial metabolism,

88 physiology, stress-responsive mechanism, carbon-energy balance, and redirecting undesirable

89 ATP sinks can improve the yield and titer of high-value metabolites (Phulara et al., 2018).

90 To meet the global energy demands and to mitigate greenhouse gases, technological

91 advancements need to focus on (i) improving the microbial cell factories to accelerate the

92 biochemical production, (ii) optimization of the existing production technologies for higher

93 efficiency, (iii) expansion of cell’s ability to produce biochemical/biofuels related designer

94 molecules that reduce CO2 emission and improve fuel quality, respectively. Integrated

95 approaches could help in overcoming the technological barriers encountered during designing

96 an efficient, effective, and reliable biofuel production pipeline (Liu et al., 2021b).

97 Genome editing has revolutionized the reconstruction efforts as it prompted metabolic

98 engineering of the native and non-native hosts for the reconstruction of metabolic pathways

99 to produce renewable biofuels. To obtain higher production titer of biofuels through strain

100 improvement, various approaches have been employed successfully including process

4
101 engineering, pathway optimization, promoter engineering, enzyme engineering, orthogonal

102 pathway synthesis, genetic modification, metabolism optimization, and competitive pathway

103 blocking (Choi et al., 2020). Advances in synthetic biology have reduced the efforts unlike

104 before in developing microbial cell factories. However, the major challenge is the limited

105 number of available obvious genes and their combinatorial impact on the metabolomic

106 system of the host organism. As in most instances, the modification results in a complex

107 genotype-phenotype relationship which leads to adverse and unintended effects (Hansen et

108 al., 2020).

109 Therefore, the key for achieving higher titers from microbial cell factories is careful

110 optimization and reconstruction of the metabolic pathway regulation (Fig. 1) in terms of

111 balanced metabolic flux, enzyme activity, and gene expression (Liao et al., 2016). This

112 review highlights the research efforts for titer improvement of alcohols, fatty acid-based

113 fuels, hydrocarbons, and isoprenoid-derived fuels and chemicals through heterologous

114 reconstruction of the prokaryotic (bacteria and cyanobacteria) and eukaryotic (yeast) cell

115 factories. It also spotlights the challenges encountered in various system biology and genetic

116 engineering approaches to produce fourth-generation biofuels and biochemicals along with

117 the possible solutions to overcome these challenges.

118

119 2. Heterologous synthesis of advanced biofuels and high-value biochemicals

120 In recent years, synthetic biology, OMICs, and system biology approaches have been

121 employed to redesign the native microbial pathways to produce the desired products in bulk

122 amounts, for example, commodity chemicals, drugs, secondary metabolites, organic acids,

123 biofuels, and other useful industrial biochemicals. However, several factors including

124 precursor supply, regulatory systems, enzyme efficiency, cost-effectiveness, toxicity, energy

5
125 content, product recovery, and environmental protection are needed to be considered while

126 developing biochemical producing microbial cell factories (Liu et al., 2021a).

127 Using engineered microbial platforms to produce high-value bioproducts is an eco-

128 friendly approach, unlike the traditional chemical synthesis. Genome sequence information,

129 well-established OMICS databases, and metabolic engineering tools facilitated the targeted

130 engineering of E. coli and S. cerevisiae in the bioindustry to manufacture alcohols, alkanes,

131 isoprenoids, and fatty acid-derived biomolecules for diverse applications. The development

132 of highly advanced genome editing techniques including MAGE/eMAGE and CRISPR/Cas9

133 can expedite the genetic engineering of conventional and non-conventional hosts to develop

134 efficient workhorses for biofuel production (Niu et al., 2018). Engineering S. cerevisiae to

135 produce bioactive compounds is relatively easy due to several reasons, i) easily tractable

136 genome over other oleaginous strains of yeast and microalgae due to the availability of

137 genetic tools for its manipulation, ii) isolation, generation, and analyses of its mutant strains

138 are easy iii) easily cultivated in defined medium, iv) fast growth rate, and v) established

139 upscaling processes. Among yeasts, Yarrowia lypolytica is reported as a suitable model

140 species to produce long-chain hydrocarbons. Other than yeast, several species of bacterial

141 strains including Corynebacterium glutamicum, Zymomonas mobilis, Lactococcus lactis,

142 Bacillus subtilis have been explored to assess their potential for biofuels production. Among

143 these, Escherichia coli has been the most popular, broadly studied, user-friendly, and easily

144 deployed host organism that holds all the genetic tools required for pathway engineering and

145 controlling the regulatory networks (Malik et al., 2018). Here, the production of various high-

146 value biochemicals along with challenges and opportunities is briefly discussed.

147

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148 2.1. Heterologous synthesis of higher alcohols

149 N-Butanol has been used as a gasoline substitute, industrial cleaner, solvents,

150 component of acrylic paints, perfumes, plastics, and polymers (Nawab et al., 2020). The

151 global market of n-butanol is increasing at a rapid rate with an estimated market value of 5

152 billion USD. Butanol is mainly produced either by chemical processes/petrochemicals (at the

153 cost of 7.0-8.0 billion USD/year) or through biological fermentation (based on the Acetone-

154 Butanol-Ethanol pathway). However, the dependence of chemical synthesis on crude oil price

155 (which vary significantly) does not make it a suitable option in the near future (Nawab et al.,

156 2020; Tanaka et al., 2020). Whereas biological synthesis of n-butanol is sustainable and is

157 considered as carbon neutral.

158 Naturally, it is produced by Clostridia spp. through Acetone-Butanol-Ethanol (ABE)

159 pathway (Fig. 2) but slow growth rate, complex nutrient requirement, by-product formation,

160 toxicity, tedious downstream processing cost, complex life cycle, and difficult genetic

161 modification limits the biological production of butanol. Furthermore, Clostridium sp. is

162 unable to directly utilize low-cost substrate such as cellulose, hemicellulose, and organic

163 waste and thus must be dependent on the molasses and starchy materials whose availability is

164 limited by geographical dependence and competition with human food respectively (Nawab

165 et al., 2020; Zheng et al., 2009). Therefore, to achieve sustainability metabolic engineering of

166 non-native hosts has been attempted because non-native hosts offer different advantages over

167 native hosts including, fast growth, solvent tolerance, use of alternative carbon sources as

168 substrate, reduced contamination risks (in case of thermophilic strains), and readily available

169 genetic tools. Hence, different engineering strategies have been adopted using non-butanol-

170 producing species (Nawab et al., 2020) such as S. cerevisiae, E. coli, Lactobacillus brevis, P.

171 putida, and B. subtilis to achieve commercial production targets of butanol hitherto (Table 1).

172 Butanol-induced toxicity is the first major hindrance to achieve commercial yields. With its

7
173 chaotropic activity of 37.4 kJ kg-1 M-1, butanol disrupts the macromolecular complexes of the

174 host cell and then causes oxidative damage which ultimately leads to product-induced growth

175 inhibition (Cray et al., 2015). To counter the toxicity issue, strategies that involve the use of

176 low temperatures or other substances with neutralizing effects against chaotropicity can be

177 considered. Different studies focused on improving butanol production through the ABE

178 fermentation pathway in Clostridium acetobutylicum. Among these the most efficient and

179 cost-effective strategies to accelerate the butanol production, are to supplement the small

180 amount of inexpensive electron receptor, Na2SO4 in the broth which changed the distributions

181 of intracellular electrons or protons, resulting in the accumulation of favorable amino acids

182 vital for the survival of host cell (Ding et al., 2018). The bacterium Staphylococcus sciuri can

183 tolerate 2.25% v/v butanol with a maximum growth of 70% reported so far (Goyal et al.,

184 2019). Keeping in view the recent progress in global R & D, butanol has the potential to

185 emerge as a sustainable, economical, and promising eco-fuel soon (Kushwaha et al., 2019).

186 Iso-butanol is another important commodity chemical having plentiful industrial

187 applications (Table 1). It has been used as a solvent for adhesives, paints, inks, coatings, and

188 many other industrial products. Furthermore, it gains popularity as a fuel additive due to its

189 applications in alkane/alkene production and transesterification/esterification reactions

190 (Moncada et al., 2017). Several research efforts have been performed to enhance isobutanol

191 production through genetic engineering. In-situ product recovery method was applied in a

192 bioreactor to attain the total titer of 50 g/L in 72 h which was 9% higher when compared to n-

193 butanol production in Clostridium using a similar system (Baez et al., 2011). Exclusion of the

194 competing pathways, cofactor imbalance resolution (Matsuda et al., 2013), and optimization

195 of excessive by-products and pathway intermediates formation in S. cerevisiae also improved

196 iso-butanol production (Milne et al., 2016). In this context, coupling of an oxidoreductive

197 pathway with mitochondria-targeted iso-butanol biosynthesis pathway was performed in S.

8
198 cerevisiae for redirecting xylose consumption from ethanol to isobutanol production. It

199 results in 23-fold improved iso-butanol production with the final titer of 2.6 g/L (Lane et al.,

200 2020). Similarly, relocalization and replacement of mitochondrial pathway by the cytosolic

201 pathway could enhance iso-butanol production by halting the competing pathway. In S.

202 cerevisiae, targeting the complete Ehrlich pathway to mitochondria and comparing the

203 overexpression of enzymes involved in the cytosolic Ehrlich pathway, the yield was

204 enhanced by 260% (Avalos et al., 2013). However, the productivity in S. cerevisiae in

205 contrast to E. coli still needs to be enhanced to achieve the commercialization of iso-butanol.

206 Overexpression of the endogenic valine synthesis pathway in Pichia pastoris enabled the

207 strain capable to produce 0.89 g/L of iso-butanol. This titer was further enhanced to 2.22 g/L

208 (43-fold increase), by applying an expression system based on an episomal plasmid with fine-

209 tuning of the bottleneck enzymes (Siripong et al., 2018). Later, the heterologous expression

210 of the ILV2 gene from S. cerevisiae in a multinuclear yeast Magnusiomyces magnusii

211 produced a stabilized transformant resulted in 620 mg/L of iso-butanol in YPD medium

212 (Kurylenko et al., 2020).

213 The 2,3-Butanediol (2,3-BDO) is the major precursor for a variety of industrial

214 products including 1,3-butadiene, solvent ketones, and gamma-butyrolactone (GBL). The

215 unique efficacy and distinct physiochemical properties of this compound make it suitable for

216 use in high-value industries like agriculture, cosmetics, and pharmaceutics. The 2,3-BDO

217 pathway was fitted into Z. mobilis along with three genes namely acetolactate synthase

218 (ALS), acetolactate decarboxylase (ALDC), and butanediol dehydrogenase (BDH) from

219 Bacillus licheniformis and Enterobacter cloacae to facilitate the conversion of pyruvate to

220 acetoin through the intermediate synthesis of α-acetolactate, and then finally to 2,3-BDO. In

221 this way, 10g/L of 2,3-BDO was produced under anaerobic conditions (Yang et al., 2017).

222 Similarly, a cyanobacterial strain, Synechocystis sp. PCC6803 was used for the heterologous

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223 synthesis of 2,3-BDO, which could be easily dehydrogenated to methyl ethyl ketone, a

224 potential fuel additive, and other important industrial solvents. A combination of

225 heterologous genes from L. lactis and B. brevis produced a final titer of 0.43 g/L (Savakis et

226 al., 2013). To produce 2,3-BDO, a combination of different genes including, ALSS from B.

227 subtilis, ALSD from E. aerogenes, and ADH from Clostridium beijerinckii were used in the

228 presence of CO2 and sunlight, and 2.38 g/L production was attained (Oliver et al., 2013).

229 Later, different strategies were adopted involving the overexpression of rate-limiting

230 enzymes catalyzing glycolysis (Yang et al., 2017) to further enhance the titer. Pilot-scale

231 production of 2,3-BDO along with 1,3-propanediol has also been achieved with the final

232 productivity of 2.2 g/L/h (Park et al., 2017). Two novel hosts Neptunomonas concharum and

233 Vibrio natriegens have been engineered to produce 2,3-BDO with high productivities (Erian

234 et al., 2020; Li et al., 2019).

235 These achievements reflect that recent breakthroughs in modern biology have paved

236 the way to develop potential strains with the capability to produce bio-based butanol, iso-

237 butanol, and 2,3-BDO to address the commercial and environmental concerns (Table 1).

238 However, detailed studies and knowledge-based strategies are required in the future for the

239 commercial production of these alcohols.

240

241 2.2. Heterologous synthesis of hydrocarbons

242 Medium-chain (C7-C13) hydrocarbons are the finest drop-in fuels and are potential

243 substitutes of diesel, kerosene, and gasoline for the transport sector while long-chain (>C22)

244 hydrocarbons are an attractive source of value-added chemicals (Liu & Nielsen, 2019).

245 Biologically, these are mostly produced through non-fermentative pathways, for example,

246 fatty acid biosynthesis pathway, valine pathway, and butanol pathway (Fig. 3). For the very

247 first time, alkanes were heterologously synthesized in E. coli by expressing the fatty acyl-

10
248 ACP/CoA reductase and the fatty aldehyde deformylating oxygenase from cyanobacteria to

249 convert fatty acid metabolism intermediates into alkanes and alkenes ranging from C13 to C17,

250 and a titer of 0.025 g/L was achieved (Schirmer et al., 2010). Afterward, a variety of FAR

251 and FADO enzymes were selected from different sources, expressed in E. coli heterologously

252 and a hydrocarbon titer of 0.58 g/L was achieved (Howard et al., 2013).

253 A novel valine synthesis pathway was assembled in E. coli strain BW25113 to

254 produce propane. The ALSS gene (acetolactate synthase from B. subtilis) was expressed to

255 convert pyruvate into 2-acetolactate which was then converted to 2-ketoisovalerate with the

256 overexpression of two genes namely ketol-acid reductoisomerase (ILVC) and dihydroxy-acid

257 dehydratase (ILVD) of E. coli. To further convert the 2-ketoisovalerate into isobutyraldehyde

258 and then to propane, alpha-ketoisovalerate decarboxylase (KIVD) gene from L. lactis and

259 aldehyde-deformylating oxygenase (ADO) was obtained from P. marinus MIT9313. At this

260 stage, 13 different types of aldehyde-reductase encoding genes namely YQHD,

261 ADHE, EUTG, ADHP, YJGB, YIAY, YAHK, FUCO, IDHA, FRDABCD, PFIB, FNR,

262 and DKGA were knocked out in E. coli because the endogenic synthesis of aldehyde

263 reductases is known to halt the propane production by switching the isobutyraldehyde

264 conversion from propane to iso-butanol (Rodriguez & Atsumi, 2014). Interestingly, knocking

265 out of these genes did not affect the growth of the mutant strain. By using two ado mutants

266 and process optimizations, a 267 μg/L propane was produced which was a three-fold increase

267 in production (Zhang et al., 2016).

268 Metabolic pathway engineering has also been employed on cyanobacteria to produce

269 hydrocarbons and other valuable chemicals. Genetically modified cyanobacteria/microalgae

270 are considered due to their environmental benefits such as wastewater treatment, CO2

271 sequestration, and nutrient/ CO2 assimilation thus offering an environment-friendly

272 biochemical production facility (Fu et al., 2021). Metabolic engineering of the Synechocystic

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273 sp. and Chlamydomonas reinhardtii was performed for the one-step conversion of CO2 into

274 hydrocarbons. For this purpose, the CAR gene was replaced with UndB of Pseudomonas, and

275 ADO was replaced with FAP from Chlorella. Engineered C. reinhardtii was able to produce

276 8-fold higher alkene (0.008 g/L) and Synechocystic could produce 19-fold higher alkane

277 (0.077 g/L) when compared to the native strain (Yunus et al., 2018). Acetyl-CoA is the major

278 intermediate to produce a multitude of products including fatty acids, ethanol, butanediol,

279 butanol, etc. However, in cyanobacteria, the flux towards acetyl-CoA is limited due to

280 pyruvate decarboxylation. Therefore, overexpression of PDH (pyruvate dehydrogenase)

281 genes in S. elongatus was performed to transfer the flux from CBB (Calvin–Benson–

282 Bassham cycle) towards acetyl-CoA. It was followed by combinatorial engineering with

283 isopropanol and acetate biosynthesis pathways. It resulted in 0.29 g/L of isopropanol and 0.59

284 g/L of acetate titer that is 9-fold and 7-fold higher than the wild-type strain (Hirokawa et al.,

285 2020). These findings highlighted the necessity of CO2 fixation for enhanced hydrocarbon

286 production and emphasized that optimizing the activity of Rubisco can further increase

287 productivity.

288 The strains of S. cerevisiae have been constructed using the alkane biosynthesis

289 pathway to produce long-chain alkanes at a commercial scale by deleting the endogenous

290 hexadecenal dehydrogenase gene (HFD1) for the HFD1 hampers the synthesis of alkanes.

291 Hence, its deletion along with the expression of the redox system can enhance the titer of

292 long-chain alkanes (Buijs et al., 2015). Conversion of free fatty acids to aldehydes was

293 catalyzed by expressing α-dioxygenase (a fatty acid) from Oryza sativa in S. cerevisiae.

294 These aldehydes were then transferred to the desired alkanes by engineering cyanobacterial

295 aldehyde deformylating oxygenase (Foo et al., 2017). As an alternative to this two-step

296 process involving aldehydes formation, one-step synthesis of alkenes through the

297 decarboxylation of fatty acids is preferred because of higher titer and yield (Zhu et al., 2017).

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298 Manipulating the fatty-acid metabolism in S. cerevisiae by the dynamic regulation of a

299 desaturase-like enzyme UndB, expressing an electron transport system, and a FATP1

300 exporter to secrete the product from the cells, resulted in the synthesis of 35.3 mg/L of 1-

301 alkenes, >80% of which was exported outside the cells (Zhou et al., 2018). To increase the

302 titer of alkane, an FDH gene from Xanthobacter sp. 91 was expressed in E. coli to catalyze

303 the conversion of formate into a reductant that can be used by ADO. This strategy proved to

304 be very helpful in maximizing the biological production of alkanes (Jaroensuk et al., 2020).

305 These studies reflected the remarkable potential of non-natural hosts for the biosynthesis of

306 long-chain hydrocarbons (Table 2) and reflect the possible strategies which can be employed

307 to harness their full potential and to achieve robustness by improving the yield and titer of the

308 final product.

309

310 2.3. Synthesis of fatty-acid-derived bioactive compounds using Yeast hosts

311 Fatty acids (FA) are renowned precursors to produce oleochemicals that have many

312 industrial applications. Free fatty acids (mainly present as triacylglycerols) cannot be

313 exploited as a transportation fuel owing to the presence of ionic carboxyl group unless they

314 are transformed into hydrophobic molecules like hydrocarbons, fatty alcohols, fatty acid alkyl

315 esters (FAEEs), and fatty acid methyl esters (FAMEs). The other FA-based compounds

316 include methyl ketones have been used as fragrance and flavoring agents, fatty alcohols have

317 applications in lubricants, laundry detergents, surfactants, personal care products, and

318 medicine production. Whereas long and medium-chain oleochemicals are used to produce

319 cosmetics, pharmaceuticals, plasticizers, pesticides, polymers, detergents, lubricants,

320 coatings, and film-processing agents (Yan & Pfleger, 2020). Odd-chain FAs are of

321 commercial importance due to their anti-microbial, anti-inflammation, and anti-allergic

13
322 properties. They are also used as disease risk detection biomarkers for coronary heart disease,

323 diabetes mellitus, and food intake assessment (Park et al., 2018).

324 Different types of heterologous pathways have been installed into S. cerevisiae to

325 synthesize free fatty acids which are easily convertible to alkanes (Fig. 3 & Table 3). To

326 enhance the biosynthesis and accumulation of TAG, three essential fatty-acid biosynthetic

327 genes encoding acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid

328 synthase 2 (FAS2) were overexpressed in S. cerevisiae. Interestingly, the engineered strain

329 accumulated 4-folds lipids when compared to the native strain. The overall production titers

330 of free fatty acids and FAEEs were up to 0.4 g/L and 0.005 g/mL, respectively (Runguphan

331 & Keasling, 2014). To improve the titer of FAs in S. cerevisiae, metabolic flux analysis was

332 done to knock out the competing enzymes. Complying with the analysis, the glycerol-3-

333 phosphate dehydrogenase gene (GPD1) was knocked out to reduce the competition of carbon

334 flux, which resulted in a 22% increase in FAs production, which was further improved to

335 56% after the overexpression of ATP citrate lyase (ACL) from Y. lipolytica (Ghosh et al.,

336 2016). The oleaginous yeast strain Y. lipolytica serves as an excellent chassis to produce

337 fatty-acid-derived fuels as it provides precursor lipids in huge amounts. To achieve an

338 enhanced biosynthesis of FAEEs, the wax ester synthase gene (WS) from Marinobacter

339 hydrocarbonoclasticus was heterologously introduced into Y. lipolytica, which enhanced the

340 extracellular FAEEs level from 0.18 g/L to 1.18 g/L when compared to the wild-type strain

341 (Gao et al., 2018). Similarly, Y. lipolytica was engineered to boost the biosynthesis of the

342 TAG. An expression system was developed by employing an intron holding translation

343 elongation factor 1-α promoter in Y. lipolytica. This expression system was used to

344 overexpress the diacylglycerol acyltransferase (DGA) in yeast cells, which enabled the yeast

345 cells to produce 33.8% lipid content of dry cell weight. By co-expressing, the DGA and

346 acetyl-CoA carboxylase, lipid accumulation in cells was enhanced to 61.7% of dry cell

14
347 weight (Tai & Stephanopoulos, 2013). To accelerate the lipid biosynthesis pathway in Y.

348 lipolytica, three genes, mammalian delta-9 stearoyl-CoA desaturase (SCD), Acetyl-CoA

349 carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) were overexpressed, and

350 lipid production was enhanced from ~ 5 g/L to ~ 55 g/L when compared to the wild-type

351 strain (Qiao et al., 2015). A different route for the synthesis of FAEEs in Y. lipolytica was

352 developed by using endogenously produced ethanol. In this catalytic route two genes, PDC1

353 and ADH1 from S. cerevisiae and a WS gene from Marinobacter hydrocarbonoclasticus were

354 engineered which enhanced the production titer of FAEEs to 360.8 mg/L (Yu et al., 2020).

355 Such studies signify the potential of yeast strains as an alternative host to produce 4G fuels

356 and other industrial chemicals.

357

358 2.4. Synthesis of fatty-acid-derived compounds using non-yeast hosts

359 In addition to yeast, microbes, and cyanobacteria exhibit a promising potential to

360 produce fatty-acid-derived bioactive compounds (Table 4), which are often termed as

361 microdiesel or microbial oil. However, strict regulation of the FA biosynthesis pathway in

362 these organisms limits its utilization. Therefore, optimization of pathway regulation is

363 required to improve the synthesis of FFA (free fatty acid) and other related products (Janßen

364 & Steinbüchel, 2014). Recently, E.coli was engineered for enhanced FA biosynthesis by

365 introducing heterologous genes from the Corynebacterium glutamicum and Pseudomonas

366 putida into E.coli. Co-expression of acetyl-CoA carboxylase (ACC) from C. glutamicum and

367 pantothenate kinase (coaA) from P. putida in E. coli, improved the FA biosynthesis by 3.1-

368 fold. Whereas, co-expression of ACC and fatty acid synthase (fasA) in E. coli from C.

369 glutamicum improved FA biosynthesis by 3.6-fold. Simultaneous expression of these three

370 genes in E. coli resulted in 5.6-fold improved FA biosynthesis with the final titer of 0.691 g/L

371 FA. The results signify that manipulation of the intracellular CoA pool is an effective strategy

15
372 to improve FA biosynthesis (Satoh et al., 2020). A combination of competing pathways with

373 multi-module optimization was explored to enhance the fatty-aldehyde balance for improved

374 alkane production. The combined strategy improved alkane production from trace amounts to

375 1.31 g/L (Cao et al., 2016b). In another study, modular pathway engineering of multigene

376 fatty-acid synthesis pathways and combinatorial optimization techniques also improved the

377 fatty-acid production in E. coli up to 8.6 g/L (Xu et al., 2013).

378 Short-chain fatty acids can also be synthesized in E. coli by exploiting the native fatty

379 acid synthesis pathway. Three strains, harboring different thioesterases; tesBF

380 from Bryantella formatexigens, tesAT from Anaerococcus tetradius, and tesBT

381 from Bacteroides thetaiotaomicron were compared to evaluate their competence for the

382 synthesis of short-chain fatty acids (butyric acid). Among all these strains, the strain

383 expressing the tesBT gene produced butyric acid with the highest titer of 1.46 g/L (Jawed et

384 al., 2016). Different metabolic engineering strategies have been attempted to engineer the

385 bacterium Rhodococcus opacus to produce FFAs, FAEEs, and long-chain hydrocarbons (Kim

386 et al., 2019a). Despite these efforts, FA and FAEE biosynthesis are yet to meet the

387 commercial levels. To understand the fatty acid metabolism in E. coli, system biology

388 approaches were practiced which elucidated that NADPH and ATP serve as bottlenecks in

389 fatty-acid production (He et al., 2014).

390 Cyanobacteria are also being engineered to produce fatty acids, but the resulting titers

391 are not encouraging (Table 4) as compared to microbes. FA production in C. reinhardtii was

392 improved by 56% without affecting the growth through overexpression of acyl-ACP

393 thioesterases (TE) from Dunaliella tertiolecta into C. reinhardtii (Tan & Lee, 2017). To

394 enhance the titer, the acyl-ACP thioesterase and acetyl-CoA carboxylase genes from C.

395 reinhardtii CC-503, were co-expressed in S. elongatus 7942, but due to the adverse effects of

396 FFA on cell physiology, the overall titer could not be improved according to the expectations

16
397 (Ruffing, 2013). In a subsequent study, the biosynthesis of fatty alcohols was enhanced in

398 Synechocystis sp. PCC 6803 by articulating two different fatty acyl-ACP reductases from

399 Marinobacter aquaeolei and the O. sativa using multiplex CRISPR-interference

400 (Kaczmarzyk et al., 2018). Later, the same strain was manipulated to produce and secrete the

401 methanol-free FAMEs by heterologous expression of thioesterase from Umbellularia

402 californica, and O-methyltransferase from Drosophila melanogaster (Yunus et al., 2020).

403 These studies (Table 4) indicated that a deep understanding of molecular networks involved

404 in fatty acid synthesis followed by a fine-tuning of the engineered strain could be exploited as

405 a promising strategy to produce FFA-derived advanced biofuels in cyanobacteria.

406 2.5. Heterologous synthesis of isoprenoids-based biofuels and biomolecules

407 Isoprenoids are a captivating group of biological compounds with over 60,000

408 reported members. They have naturally been produced in plants and bacteria through MEP

409 (2-C-methyl-D-erythritol 4-phosphate) pathway and MVP (mevalonate) pathway in

410 eukaryotes and archaea. These pathways produce monoterpenes (C10), sesquiterpene (C15),

411 diterpene (C20), sesterterpene (C25), triterpene (C30), and tetraterpene (C40) isoprenoids

412 (Daletos et al., 2020b). They have been an excellent source of biodiesel, jet fuels, and fuel

413 additives because their rings and branching patterns bestow them a higher-octane number,

414 water immiscibility, high energy density, and low burning point (Walls & Rios-Solis, 2020).

415 These molecules do not require a post-transesterification process and do not undergo

416 premature ignition in the internal combustion engine. Moreover, these organic compounds

417 have been used as a fragrance component in perfumes and coloring/flavoring agents in the

418 food industry. They are also important to produce commodity chemicals, plant oils, bio-

419 pesticides, and pharmaceutical drugs (Rodrigues & Lindberg, 2021). However, their natural

420 host could accumulate these compounds only in minute quantities which are unable to fulfill

421 the commercial demand of these biochemicals. Therefore, heterologous production of

17
422 isoprenoids through metabolic pathway engineering (Fig. 3) is employed in microbial cell

423 factories for functional and chemical characterization of these compounds in higher quantities

424 (Li & Wang, 2016).

425 Natural hosts including yeast, bacteria, and cyanobacteria were genetically modified

426 to enhance isoprenoid production (Table 5). S. cerevisiae was introduced with geraniol

427 synthase from Ocimum basilicum to produce geraniol (monoterpene). Moreover, the

428 expression of heterologous geraniol synthase in the mutant strain (disrupted with farnesyl

429 diphosphate) enhanced geraniol synthesis. Later, a 5 mg/L increase in geraniol production

430 was attained in the engineered strain when compared to the previously achieved titers in E.

431 coli and S. cerevisiae (Fischer et al., 2011). In another study, S. cerevisiae∆ERG20 mutant

432 strain AE9 K197G was engineered with limonene synthase from Perilla frutescens and

433 limonene from Citrus limon to catalyze the geranyl diphosphate (monoterpene precursor)

434 synthesis in host cells. Although, the mutant strain synthesized limonene in higher amounts

435 (Jongedijk et al., 2015), these high levels of limonene secretion impaired the growth of host

436 cells when concentration reached 0.5-0.8g/L. Similarly, a synthetic pathway was constructed

437 in Y. lypolytica involving the heterologous expression of limonene synthase (LS) and neryl

438 diphosphate synthase 1 (NDPS1) to produce limonene which produced a final titer of

439 23.56 mg/L (Cao et al., 2016a). Later, limonene synthases from two different plants namely

440 C. limon and Mentha spicata were introduced in the Synechocystis 6803 for enhanced

441 limonene synthesis (Lin et al., 2017). The endoplasmic reticulum of S. cerevisiae was

442 extended by overexpressing the INO2 gene (this gene regulates the size of ER) which

443 improved the biosynthesis of squalene by a factor of 71 with a final titer of 634 mg/L (Kim et

444 al., 2019b). Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA

445 reductase of mevalonate and prenyl diphosphate pathways, the synthesis of farnesol,

446 geranylgeraniol, and nerolidol was enhanced in S. cerevisiae (Ohto et al., 2009). The

18
447 mevalonate pathway of E. coli was engineered to synthesize other high-octane fuels including

448 3-methyl-2-butenol, 3-methyl-3-butenol, and 3-methyl butanol from isopentenyl diphosphate

449 (IPP) (Chou & Keasling, 2012).

450 Direct conversion of CO2 into isoprene biosynthesis has been attempted in genetically

451 modified S. elongatus where heterologous expression of the mevalonate pathway followed by

452 fine-tuning through metabolite profiling and dynamic flux analysis resulted in 1.26 g/L of

453 isoprene production (Gao et al., 2016). Amorphadiene is a sesquiterpenoid that can be used as

454 biofuel and antimalarial agents. Engineering of the MEP pathway along with amorphadiene

455 synthase in S. elongatus resulted in a 23-fold increase in the amorphadiene production when

456 compared to the parent strain (Choi et al., 2016). The model host C. reinhardtii was also

457 engineered to produce non-native diterpenoids through the heterologous expression of

458 diterpene synthases and enzymes involved in the MEP pathway (Lauersen et al., 2018). The

459 mixture of isoprenoid alcohols C5-C15 (isopentenol, geraniol, and farnesol) was produced

460 (1652 mg/L) in the E. coli by introducing the heterologous MVP pathway and overexpressing

461 two genes: NUDB (dihydroneopterin triphosphate diphosphohydrolase) and ISPA (FPP

462 synthase) using glycerol as a substrate (Zada et al., 2018). In another study, a two-fold

463 increase (2.1 mg/L) in the isopentenol production in B. subtilis was observed in comparison

464 to the wild-type strain after the overexpression of NUDF (encoding ADP-ribose

465 pyrophosphatase) and DXS (1-deoxy-D-xylulose-5-phosphate synthase) genes (Phulara et al.,

466 2018). This titer was further increased to 6 mg/L by modulating the physicochemical factors

467 (Phulara et al., 2019). Recently, Synechocystic sp. was engineered by heterologously

468 expressing bisabolene synthase (AGB) and farnesyl-pyrophosphate (ISPA) genes along with

469 other bottleneck enzymes (IDI and DXP) of the native MEP pathway to increase carbon flow

470 towards bisabolene biosynthesis. 0.18 g/L of bisabolene production was achieved by growing

471 modified strain in a high-density cultivation system (Rodrigues & Lindberg, 2021).

19
472 Although, genetic engineering strategies are being extensively applied to produce a

473 high level of isoprenoids, yet heterologous synthesis of these secondary metabolites causes

474 several problems. Most importantly, owing to their hydrophobic nature, higher titers of the

475 isoprenoids are toxic, as they disassemble the membrane integrity by interacting with the

476 mitochondrial membrane. There is a need to design improved molecular designs to deal with

477 the toxicity issues in the future. In this regard, the possible solutions include tolerance

478 engineering, the use of secreting systems instead of accumulating systems, co-culture

479 systems, the introduction of unique by-pass pathways for isoprenoids, increased lipid

480 production, and the development of cell-free systems to alleviate the effects of cellular

481 toxicity as reviewed previously in detail (Daletos et al., 2020a; Malik et al., 2021; Sindhu et

482 al., 2021).

483

484 3. Economic and environmental aspects of producing bio-commodities through

485 microbial platforms

486 Successful transition from laboratory to commercial scale requires a detailed techno-

487 economic evaluation. The selection of appropriate feedstock and its pretreatment to release

488 the desired substrate are the major contributing factors in the cost. It has been reported that

489 considering poplar as feedstock and ionic-liquid for the pretreatment the selling cost of the

490 lignocellulosic ethanol would be around $4.5/gallon. Whereas utilization of algae as a

491 feedstock for bioethanol production would cost $3.2/gallon to $2.95/gallon depending upon

492 the production scale and the processing route (Daletos et al., 2020a). Butanol production cost

493 at $1.8/L which could be further reduced to $0.6/L depending upon the substrate, production

494 route, and recovery cost (Phulara et al., 2019). Another study which was focused on

495 metabolic engineering-based isoprenoid production reported cost reduction from $465/kg

496 limonene to $2.02/kg limonene by following the scheme which yields 95% of limonene from

20
497 24% glucose concentration instead of the case which yield 0.45% of limonene from 14%

498 glucose concentration. Two different scenarios based on metabolic engineering and solvent

499 evaporation were evaluated for toxicity reduction which indicated that solvent evaporation is

500 7% more expensive than the metabolic engineering approach (Walls & Rios-Solis, 2020).

501 Therefore, focusing on the biorefinery approach to obtain high-value byproducts in addition

502 to the desired product could help to reduce the final production cost.

503 Environmental analyses have shown that biobased chemicals and biofuels production

504 also offers environmental security as it reduces the emission of hazardous and toxic gases

505 such as sulfur oxides (SOx), nitrogen oxides (NOx), and carbon monoxide (CO) and thus

506 reduces the chances of respiratory problems, cancer, and other pollution-related diseases.

507 Fourth-generation biofuels and biochemicals are also considered environmentally friendly

508 due to the recirculation of CO2 into bioproducts thus helping to mitigate global warming.

509 Moreover, they usually do not produce toxic compounds and are biodegradable in nature. It is

510 estimated that biofuel production reduces greenhouse gas emissions by 50% (Tarafdar et al.,

511 2021). However, there is a need to focus on risk management to reduce the environmental

512 leaks of the genetically modified microbes which may pose a harmful impact on the

513 environment and ecosystem by horizontal gene transfer, causing toxicity, and changing

514 natural habitat (Fu et al., 2021). Though efforts had been made to address these issues at

515 industrial scale yet it requires higher capital costs which reduces the cost-effectiveness of

516 biofuel/biochemical production.

517 4. Conclusion

518 Bioengineering of microbial hosts for the heterologous synthesis of fourth-generation

519 biofuels and biochemicals offers promising opportunities. Diversification in the spectrum of

520 native and non-native hosts can further expand the understanding of metabolic pathways for

521 their exploitation in the heterologous systems. Improvement of metabolic pathways, fine-

21
522 tuning of downstream processing, development of new metabolic tools, and synthetic biology

523 approaches for robust microbial strains development would provide all necessary genetic

524 elements to build up an efficient assembly line for enhanced bioproduction. However, further

525 research activities are required to compete at the market of fossil-derived products in terms of

526 their efficacy and cost-effectiveness.

527

528 CRediT authorship contribution statement

529 Muhammad Aamer Mehmood: Conceptualization, Writing - review & editing, Ayesha

530 Shahid: Writing - original draft. Sana Malik: Writing - original draft. Ning Wang: Writing -

531 original draft. Muhammad Rizwan Javed: Writing - original draft. Muhammad Nabeel

532 Haider: Writing - original draft. Pradeep Verma: Writing - review & editing. Muhammad

533 Umer Farooq Ashraf: Writing - review & editing. Nida Habib: Writing - review & editing.

534 Achmad Syafiuddin: Conceptualization, Writing - review & editing. Raj Boopathy:

535 Conceptualization, Writing - review & editing.

536

537 Acknowledgments

538 The authors are thankful to the Higher Education Commission, Pakistan (HEC Project

539 NO. NRPU-7300) for their financial support. The collaborative effort from Sichuan

540 University of Science and Engineering, Government College University Faisalabad,

541 Universitas Nahdlatul Ulama Surabaya, Nicholls State University, and the Central University

542 of Rajasthan is highly appreciated.

543

22
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885 yield of monoterpenes and sesquiterpenes. Tree Physiology.
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887 microbial-based processes for terpenes production. Biotechnology for Biofuels. 11(1),
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900 butanediol production from lignocellulosic biomass sugars. Biotechnology for
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920 hydrocarbon fuel. Metabolic Engineering. 49, 201-211.
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939 Engineering. 44, 81-88.
940
941

34
942

943

944

945 Fig. 1. Different steps towards strain improvement through metabolic engineering (native

946 and non-native) to produce advanced biobased fuels chemicals.

947

35
948

949 Fig. 2. Genetically modified ABE, butanediol biosynthesis, and keto-acid biosynthesis

950 pathways for the improved production of subsequent end-product. 235-fold improved butanol

951 production (4.96 g/L) was reached in Clostridium by overexpression of TER (trans-enoyl-

952 coenzyme A reductase gene) in combination with the introduction of an acid-reassimilation

953 pathway to direct the carbon flow towards butyrl-CoA (Wen et al., 2020). Modified S.

954 cerevisiae with overexpressed SFA1 (alcohol dehydrogenase) gene was able to yield 0.492 g/g

955 of ethanol with different lignocellulosic hydrolysates (Zhu et al., 2020). 0.41 g/L of acetone

956 was obtained by introducing AAM bypass (the combination of acetate-acetylCoA (AA) bypass

957 and artificial M-module) in Synechococcus. The M-module consists of phosphoenolpyruvate

958 synthetase (PPS), phosphoenolpyruvate carboxylase (PPC) and methyl malonyl‐CoA

959 carboxyltransferase (MMC) rearranges carbon from pyruvate to acetyl-CoA while AA bypass

960 overcome rate-limiting step of pyruvate dehydrogenase complex (PDC) (Lee et al., 2020).

961 Genetically modified Clostridium with overexpressed sol-operon and EC cassette improves the

962 ABE production yield to 8%, 18% & 12% respectively with a final titer of 15.4 g/L (Wang et

36
963 al., 2019). Engineered E. coli strain with activated EDP (Entner–Doudoroff) pathway, glucose

964 inducible system (HexR-Pzwf1), and deleted side-pathways was able to produce 71% improved

965 2,3-butanediol with a final titer of 11 g/L (Sathesh-Prabu et al., 2020). Inactivation of STHA

966 (soluble transhydrogenase), overexpression of ILVC and ILVD, and introduction ALSS

967 (feedback‐resistant acetolactate synthase), KIVD (ketoacid decarboxylase), and YQHD

968 (aldehyde dehydrogenase) resulted in 22 mg/g of iso-butanol production in P. putida (Nitschel

969 et al., 2020).

970

37
Fig. 3. Genetic modifications in the isoprenoid biosynthesis pathway (MER/MVP),

hydrocarbon biosynthesis, and fatty-acid pathways for the improved synthesis of subsequent

end-product. Overexpression of HMGS for metabolic exchange between MEP and MVP

pathways results in 2-4 times improved monoterpenes and sesquiterpenes production

respectively (Wu et al., 2020). Heterologous expression of NDPS1 and TLS along with the

overexpression of ERG20 and it’s variant in S. cerevisiae yield 9.8 mg/L of limonene (Hu et

al., 2020b). High-yielding yeast with GPP chassis and miltiradiene synthase chimeric (TPS1

and KSL1) yielded 3.5 g/L miltiradiene in 5-L fermenter (Hu et al., 2020a). Construction of the

1-undecene pathway in ferulate-tolerant Acinetobacter was achieved through the expression of

TESA and UNDA. The engineered strain produced 694 µg/L of 1-undecene (Luo et al., 2019).

Recently, the introduction of ALKS, YFDX, and YFDR in microbe have been suggested as an

efficient strategy for 389% improved alkane production (Mori, 2020). Improvement in the lipid

pool of Yarrowia by knock-in and knock-out of key genes and targeting it to subcellular

compartmentalization improved TAG by 51% resulting in 1.64 g/L of FAME production (Yang

et al., 2019). Similarly, introducing wax synthase genes in Yarrowia in combination with the

38
reconstitution of the ethanol pathway (knock-in PDC and ADHB) and deletion of competitive

pathways improved the FAEE production by 24-folds (Ng et al., 2020).

39
Table 1. Overview of heterologous synthesis of alcohols-based biofuels and their challenges.

Advanced
Host Targeted gene/pathway Strategy Challenges References
biofuel (titer)

Butanol Clostridium 6-phosphofructokinase Double The toxicity of butanol decreased (Ventura

(19.12 g/L) acetobutylicum (PFKA) and pyruvate kinase overexpression growth rate and cell density et al., 2013)

(PYKA)

Butanol Clostridium Heat shock protein Overexpression Cell growth inhibition with (Fu et al.,

(12.15 g/L) tyrobutyricum increased titers of butanol 2021)

Iso-butanol P. pastoris 2-keto acid degradation Overexpression Over-producing KIV decreases the (Siripong et

(2.22 g/L) pathway production of other alcohols al., 2018)

40
Iso-butanol S. cerevisiae Cytosolic isobutanol Blocking undesirable Competition between isobutanol (Wess et

(2.09 g/L) synthesis pathway pathways and and other pathways to produce al., 2019)

overexpression other metabolites might be

limiting factor for increased level

of iso-butanol

2,3- butanediol Z. mobilis ALS, ALDC, BDH genes Genetic engineering The ratio of the cellular (Yang et al.,

(10 g/L) NADH/NAD+ was disturbed 2016)

possibly due to inhibitors, and

resulted in slow growth

2,3-butanediol Synechococcus Acetoin Biosynthetic Strain engineering Acute toxicity (Oliver et al.,

(2.38 g/L) elongatus Pathway 2013)

1,3-PD (42.7 Klebsiella LDHA gene Genetic engineering Cell growth retardation (Park et al.,

g/L), 2,3-BD pneumoniae 2017)

(1.8 g/L)

41
2,3-BDO Pichia Pastoris ALSS and ALSD Genetic engineering Prohibited cell growth due to (Yang &

(45 g/L) accumulation of acetaldehyde in Zhang, 2018)

the cells

Isopentanol S. cerevisiae LEU1, LEU2, and LEU4 Mitochondrial Controlling the export of (Hammer et

(1.24 g/L) compartmentalization intermediates from mitochondria al., 2020)

and unidentified mitochondrial

carriers

42
 Table 2. Advances and challenges in the heterologous synthesis of hydrocarbon-derived biofuels and biochemicals

Advanced biofuel (titer) Strain/ Host Targeted gene/pathway Strategy Challenges References

Isobutyraldehyde (35 g/L) E. coli ADHP, EUTG, YIAY, Deletion of YQHD Combined deletion of genes (Rodriguez

YJGB, BETA, FUCO, caused decreased iso-butanol & Atsumi,

EUTE. production 2012)

Alkane E. coli Deletion of endogenous Multi-modular Endogenic formation fatty (Cao et al.,

(1.3 g/L) aldehyde reductase optimization alcohols thought to be 2016b)

(AHER), expression of competitive with alkane

fatty alcohol oxidase and production

ensuing transcriptional

factors of AAR, ADHP

and ado genes

Heptadecane (10.2 mg/l) A. carbonarius Alkane biosynthesis Heterologous An unknown, innate fatty (Sinha et al.,

Pentadecane (2.7 mg/l) pathway expression aldehyde dehydrogenase 2017)

networks diverted the fatty

43
 aldehydes back to the fatty acid

metabolism

Undecanal (406.2 μg/L) S. cerevisiae α‐dioxygenase Whole cell Alternate of cADO should be (Foo et al.,

Tridecanal (692.0 μg/L), biocatalytic considered for the efficient 2017)

Pentadecanal (2,332.9 μg/L), conversion swap over of aldehydes to

Heptadecanal (280.7 μg/L) alkanes

Alkenes (1.48 g/L) Cupriavidus Ferredoxin NADP Overexpression The expression of a (Crépin et

necator reductase ferredoxin-ferredoxin-NADP+ al., 2018)

reductase system sharply

lowered the C-flow towards

fatty aldehydes.

Fatty alcohols (5.8 g/L) MhFAR Heterologous Intracellular accumulation (Cordova et

expression al., 2020)

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Table 3. Overview of strategies, challenges, in producing fatty-acid derived biochemicals and biofuels using Yeast as microbial cell factory

Advanced Targeted
Strain/host Strategy Challenges References
biofuel (titer) gene/pathway

Need to enhance production of

FAEEs Yarrowia PDC1 and ADH1, chromosome‐based (Yu et al.,
FAEEs to reach commercially
0.36 g/L lipolytica GAPDH co‐overexpression 2020)
acceptable level

Toxicity assessment and (Tai &

Odd-chain FA Deletion and flux-
Y. lipolytica PDH1 understanding the role of propionic Stephanopou
0.57 g/L diversion
catabolism in odd FA production los, 2013)

fatty acids produced showed

Oleic acid Rhodosporidium Delta-9 fatty acid (Tsai et al.,
OLE1 promising potential to be blended
2.5 g/L toruloides desaturase overexpression 2019)
with vegetable oils.

45
 FA activation

genes, Creating higher titers of FAs require

FFAs phosphatidate Gene deletion for flux- substantial amounts of acetyl-CoA, (Ferreira et
S. cerevisiae
0.102 g/g phosphatase genes, redirection ATP and NADPH, making it difficult al., 2018)

storage lipid to engineer

formation genes

46
Table 4. Overview of fatty-acid derived biochemicals production from non-yeast microbes, their pathways, and limitations.

Advanced Targeted
Strain/host Strategy Challenges References
biofuel (titer) gene/pathway

Require understanding and

Metabolic engineer the regulatory (Liu et al.,

Fatty alcohols E. coli fatty acyl-ACP
engineering strategies interactions between 2014)

biosynthetic intermediates

Butyric acid Bacteroides P1 phage (Jawed et al.,

TESBT -
1.46 g/L thetaiotaomicron transduction method 2016)

FFAs 50.2 g/L

FAEEs 21.3 g/L Metabolic

Rhodococcus Overexpression (Kim et al.,
long-chain engineering -
opacus (acyl-CoA) 2019a)
hydrocarbon strategies

5.2 g/L

47
 Requires further
FFA Synechococcus sp. Overexpression of (Ruffing,
RuBisCO investigation and
0.13 g/L PCC 7002 genes 2013)
development

Omega-3 FA
Δ6-desaturase Heterologous (Yoshino et
0.12 g/L Synechococcus sp. -
(Δ6Des) overexpression al., 2017)

TAGs Synechocystis sp. ATFA encoding Synechocystis sp. (Tanaka et

Lower productivity
(0.51 nmol/mL) PCC6803 WS/DGAT PCC6803 al., 2020)

FAME Synechocystis sp. Fatty Acid Methyl Heterologous (Kang et al.,

Inhibited cell growth
36.98-84.6 μg/g PCC6803 Ester Transferase expression 2021)

48
Table 5. Overview of heterologous synthesis of isoprenoid-based biochemicals in non-yeast microbial platforms

Advanced biofuel
Strain/host Targeted gene/pathway Strategy Challenges References
(titer)

Sesquiterpenoids Rhodobacter Patchoulol, CnVS, valencene Heterologous Low terpenoid precursor (Troost et al.,

0.024 g/L capsulatus synthases expression supply 2019)

HMG-CoA reductase
Squalene Require better (Tang et al.,
Y. lipolytica (HMG1) & diacylglycerol Co-overexpression
0.73 g/L manipulation strategies 2021)
acyltranferase (DGA1)

Lycopene Lycopene expression pathway Chromosomal (Hussain et

E. coli -
1.22 g/L & MEP pathway integration al., 2021)

Site-directed mutation
Limonene Mevalonate synthase EfmvaE (Wu et al.,
E. coli & ribosome-binding -
1.29 g/L & EfmvaSA110G 2019)
site engineering

49
 Heterologous Challenging optimization
Isoprene methylerythritol phosphate (Gao et al.,
S. elongatus expression of the of the MEP
1.26 g/L (MEP) 2016)
mevalonate pathway pathway flux

CRediT authorship contribution statement

Muhammad Aamer Mehmood: Conceptualization, Writing - review & editing, Ayesha Shahid: Writing - original draft. Sana Malik:

Writing - original draft. Ning Wang: Writing - original draft. Muhammad Rizwan Javed: Writing - original draft. Muhammad Nabeel

Haider: Writing - original draft. Pradeep Verma: Writing - review & editing. Muhammad Umer Farooq Ashraf: Writing - review &

editing. Nida Habib: Writing - review & editing. Achmad Syafiuddin: Conceptualization, Writing - review & editing. Raj Boopathy:

Conceptualization, Writing - review & editing.

Highlights

 Advances in metabolic engineering offer remarkable opportunities in strain development

 Bioengineered microbial platforms offer advantages of higher yields
 Engineered strains can produce fourth-generation biofuels and biochemicals
 However, efficient product recovery needs future research

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