Developed and emerging 1,4-butanediol commercial production strategies: forecasting the current status and future possibility.

1,4-Butanediol (1,4-BDO) is a valuable industrial chemical that is primarily produced via several energy-intensive petrochemical processes based on fossil-based raw materials, leading to issues related to: non-renewability, environmental contamination, and high production costs. 1,4-BDO is used in many chemical reactions to develop a variety of useful, valuable products, such as: polyurethane, Spandex intermediates, and polyvinyl pyrrolidone (PVP), a water-soluble polymer with numerous personal care and pharmaceutical uses. In recent years, to satisfy the growing need for 1,4-BDO, there has been a major shift in focus to sustainable bioproduction via microorganisms using: recombinant strains, metabolic engineering, synthetic biology, enzyme engineering, bioinformatics, and artificial intelligence-guided algorithms. This article discusses the current status of the development of: various chemical and biological production techniques for 1,4-BDO, advances in biological pathways for 1,4-BDO biosynthesis, prospects for future production strategies, and the difficulties associated with environmentally friendly and bio-based commercial production strategies.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

Constructing multi-enzymatic cascade reactions for selective production of 6-bromoindirubin from tryptophan in Escherichia coli.

6-Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin-derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site-specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6-halogenase fused with flavin reductase Fre (Fre-L3-SttH), tryptophanase (TnaA), toluene 4-monooxygenase (PmT4MO), and flavin-containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6-bromoindole with 45% yield to produce 6-bromo-2-oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen.

Microbial synthesis of violacein pigment and its potential applications.

Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.

Bacillus subtilis as a robust host for biochemical production utilizing biomass.

Bacillus subtilis is regarded as a suitable host for biochemical production owing to its excellent growth and bioresource utilization characteristics. In addition, the distinct endogenous metabolic pathways and the suitability of the heterologous pathways have made B. subtilis a robust and promising host for producing biochemicals, such as: bioalcohols; bioorganic acids (lactic acids, α-ketoglutaric acid, and γ-aminobutyric acid); biopolymers (poly(γ-glutamic acid, polyhydroxyalkanoates (PHA), and polysaccharides and monosaccharides (N-acetylglucosamine, xylooligosaccharides, and hyaluronic acid)); and bioflocculants. Also for producing oligopeptides and functional peptides, owing to its efficient protein secretion system. Several metabolic and genetic engineering techniques, such as target gene overexpression and inactivation of bypass pathways, have led to the improvement in production titers and product selectivity. In this review article, recent progress in the utilization of robust B. subtilis-based host systems for biomass conversion and biochemical production has been highlighted, and the prospects of such host systems are suggested.

Microbial Production of Melanin Pigments from Caffeic Acid and L-tyrosine Using Streptomyces glaucescens and FCS-ECH-Expressing Escherichia coli.

In this study, synthetic allomelanin was prepared from wild-type Streptomyces glaucescens and recombinant Escherichia coli BL21(DE3) strains. S. glaucescens could produce 125.25 ± 6.01 mg/L of melanin with a supply of 5 mM caffeic acid within 144 h. The ABTS radical scavenging capacity of S. glaucescens melanin was determined to be approximately 7.89 mg/mL of IC50 value, which was comparable to L-tyrosine-based eumelanin. The isolated melanin was used in cotton fabric dyeing, and the effect of copper ions, laccase enzyme treatment, and the dyeing cycle on dyeing performance was investigated. Interestingly, dyeing fastness was greatly improved upon treatment with the laccase enzyme during the cotton dyeing process. Besides, the supply of C5-diamine, which was reported to lead to more complex crosslinking between melanin units, to caffeic acid-based melanin synthesis was also investigated for higher production and novel functionalities. To facilitate the supply of caffeic acid and C5-diamine, E. coli strains expressing each or combinations of tyrosine ammonia lyase/p-coumarate 3-hydroxylase, feruloyl-CoA synthetase/enoyl-CoA hydratase/aldolase, and tyrosinase/lysine decarboxylase enzymes were prepared and investigated for their eumelanin, C5-diamine, and allomelanin production from L-tyrosine and L-lysine, respectively. Finally, H-NMR, FT-IR, and MALDI-TOF analysis of the synthetic melanin pigments were attempted to obtain the chemical information.

Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants.

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l-tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.

Enhanced tolerance to inhibitors of Escherichia coli by heterologous expression of cyclopropane-fatty acid-acyl-phospholipid synthase (cfa) from Halomonas socia.

Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli.

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.

Expression, purification and characterization of halophilic protease Pph_Pro1 cloned from Pseudoalteromonas phenolica.

In this study, protease Pph_Pro1 from Pseudoalteromonas phenolica, possessing extracellular proteolytic activity and salt tolerance, was investigated for cloning, expression, and purification purposes. Through optimization, it was determined that optimum soluble recombinant expression was achieved when Pph_Pro1 was co-expressed with the pTf16 vector chaperone in LB medium supplemented with CaCl2. Pph_Pro1 was purified using osmotic shock and immobilized metal-affinity chromatography (IMAC). Isolated Pph_Pro1 activity was measured as 0.44 U/mg using casein as a substrate. Interestingly, Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostable properties. Furthermore, it was resistant to several hydrophilic and hydrophobic organic solvents. Substrate specificity and kinetic values such as Km and Vmax were determined with casein, bovine serum albumin (BSA), and algal waste protein as substrates, indicating that the Pph_Pro1 protease enzyme had a greater affinity for casein. Based on the remarkable characteristics of this Pph_Pro1 protease enzyme, it can potentially be utilized in many biotechnological industries.

Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid.

CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.

Polymeric solvent engineering for gram/liter scale production of a water-insoluble isoflavone derivative, (S)-equol.

A potent phytoestrogen, (S)-equol, is a promising isoflavone derivative drawing our great attention owing to its various biological and clinical benefits. Through selective activation of the estrogen receptor ERß or androgen receptor, (S)-equol reduces menopausal symptoms, osteoporosis, skin aging, hair loss, and incidence of prostate or ovarian cancers without adverse effects. Traditional biosynthesis of (S)-equol exploited non-productive natural equol-producing anaerobic bacteria that mainly belong to Coriobacteriaceae isolated from human intestine. Recently, we developed a recombinant Escherichia coli strain which could convert daidzein into (S)-equol effectively under an aerobic condition. However, the yield was limited up to about the 200 mg/L level due to unknown reasons. In this study, we identified that the bottleneck of the limited production was the low solubility of isoflavone (i.e., 2.4 mg/L) in the reaction medium. In order to solve the solubility problem without harmful effect to the whole-cell catalyst, we applied commercial hydrophilic polymers (HPs) and a polar aprotic co-solvent in the reaction medium. Among the examined water-soluble polymers, polyvinylpyrrolidone (PVP)-40k was verified as the most promising supplement which increased daidzein solubility by 40 times and (S)-equol yield up to 1.22 g/L, the highest ever reported and the first g/L level biotransformation. Furthermore, PVP-40k was verified to significantly increase the solubilities of other water-insoluble natural polyphenols in aqueous solution. We suggest that addition of both HP and polar aprotic solvent in the reaction mixture is a powerful alternative to enhance production of polyphenolic chemicals rather than screening appropriate organic solvents for whole-cell catalysis of polyphenols.

Enhanced ammonium removal efficiency by ion exchange process of synthetic zeolite after Na+ and heat pretreatment.

In this study, the optimum ammonium removal by activation of synthetic zeolite in the aqueous phase was investigated by batch ion exchange adsorption assay, and its surface changes due to activation modification was elucidated accordingly. Among the adsorbents examined, modified synthetic zeolite A-4 was the most effective at ammonium removal. The best activation condition of zeolite A-4 was established by Na+ and 300 °C heat treatment at pH around 6 to 7. Besides, the removal efficiency was investigated under various reaction conditions of pH, adsorbent dosage, stirring speed, and initial ammonium concentration. Finally, the adsorptive capacity Qe of synthetic zeolite A-4 activated by Na+ and heat treatment was determined as 31.9 mg/g at 1,000 mg-N/L of ammonium, whereas that of natural zeolite was measured as 16.0 mg/g. The obtained adsorption data was fitted to both Langmuir and Freundlich isotherm models, and the Langmuir isotherm model provided a better correspondence than the Freundlich isotherm. Finally, regeneration cycles for synthetic zeolite A-4 was determined for further industrial applications and efficient ammonium removal.

Production of p-hydroxybenzoic acid from p-coumaric acid by Burkholderia glumae BGR1.

p-Coumaric acid (pCA) is abundant in biomass with low lignin content, such as straw and stubble from rye, wheat, and barley. pCA can be isolated from biomass and used for the synthesis of aromatic hydrocarbons. Here, we report engineering of the natural pathway for conversion of pCA into p-hydroxybenzoic acid (pHBA) to increase the amount of pHBA that accumulates more than 100-fold. Burkholderia glumae strain BGR1 (BGR1) grows efficiently on pCA as a sole carbon source via a CoA-dependent non-ß-oxidation pathway. This pathway removes two carbons from pCA as acetyl-CoA yielding p-hydroxybenzaldehyde and subsequently oxidizes it to pHBA. To increase the amount of accumulated pHBA in BGR1, we first deleted two genes encoding enzymes that degrade pHBA in the ß-ketoadipate pathway. At 10 mM of pCA, the double deletion mutant BGR1_PB4 (Δphb3hΔbcl) accumulated pHBA with 95% conversion, while the control BGR1 accumulated only with 11.2% conversion. When a packed bed reactor containing immobilized BGR1_PB4 cells was operated at a dilution rate 0.2 h(-1) , the productivity of pHBA was achieved at 9.27 mg/L/h for 134 h. However, in a batch reactor at 20 mM pCA, growth of BGR1_PB4 was strongly inhibited, resulting in a low conversion of 19.3%. To further increase the amount of accumulated pCA, we identified the first enzyme in the pathway, p-hydroxcinnmaoyl-CoA synthetase II (phcs II), as the rate-limiting enzyme. Over expression of phcs II using a Palk promoter in a batch reaction at 20 mM of pCA yielded 99.0% conversion to pHBA, which is the highest concentration of pHBA ever reported using a biological process. Biotechnol. Bioeng. 2016;113: 1493-1503. © 2015 Wiley Periodicals, Inc.

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo.

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural.

Furfural is a toxic by-product formulated from pretreatment processes of lignocellulosic biomass. In order to utilize the lignocellulosic biomass on isobutanol production, inhibitory effect of the furfural on isobutanol production was investigated and combinatorial application of two oxidoreductases, FucO and YqhD, was suggested as an alternative strategy. Furfural decreased cell growth and isobutanol production when only YqhD or FucO was employed as an isobutyraldehyde oxidoreductase. However, combinatorial overexpression of FucO and YqhD could overcome the inhibitory effect of furfural giving higher isobutanol production by 110% compared with overexpression of YqhD. The combinatorial oxidoreductases increased furfural detoxification rate 2.1-fold and also accelerated glucose consumption 1.4-fold. When it compares to another known system increasing furfural tolerance, membrane-bound transhydrogenase (pntAB), the combinatorial aldehyde oxidoreductases were better on cell growth and production. Thus, to control oxidoreductases is important to produce isobutanol using furfural-containing biomass and the combinatorial overexpression of FucO and YqhD can be an alternative strategy.

The production of ω-hydroxy palmitic acid using fatty acid metabolism and cofactor optimization in Escherichia coli.

Hydroxylated fatty acids (HFAs) are used as important precursors for bulk and fine chemicals in the chemical industry. Here, to overproduce long-chain (C16-C18) fatty acids and hydroxy fatty acid, their biosynthetic pathways including thioesterase (Lreu_0335) from Lactobacillus reuteri DSM20016, ß-hydroxyacyl-ACP dehydratase (fabZ) from Escherichia coli, and a P450 system (i.e., CYP153A from Marinobacter aquaeolei VT8 and camA/camB from Pseudomonas putida ATCC17453) were overexpressed. Acyl-CoA synthase (fadD) involved in fatty acid degradation by ß-oxidation was also deleted in E. coli BW25113. The engineered E. coli FFA4 strain without the P450 system could produce 503.0 mg/l of palmitic (C16) and 508.4 mg/l of stearic (C18) acids, of which the amounts are ca. 1.6- and 2.3-fold higher than those of the wild type. On the other hand, the E. coli HFA4 strain including the P450 system for ω-hydroxylation could produce 211.7 mg/l of ω-hydroxy palmitic acid, which was 42.1 ± 0.1 % of the generated palmitic acid, indicating that the hydroxylation reaction was the rate-determining step for the HFA production. For the maximum production of ω-hydroxy palmitic acid, NADH, i.e., an essential cofactor for P450 reaction, was overproduced by the integration of NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii into E. coli chromosome and the deletion of alcohol dehydrogenase (ADH). Finally, the NADH-level-optimized E. coli strain produced 610 mg/l of ω-hydroxy palmitic acid (ω-HPA), which was almost a threefold increase in its yield compared to the same strain without NADH overproduction.

Isobutanol production from an engineered Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is one of the most well-known metal-reducing bacteria and it has been extensively studied for microbial fuel cell and bioremediation aspects. In this study, we have examined S. oneidensis MR-1 as an isobutanol-producing host by assessing three key factors such as isobutanol synthetic genes, carbon sources, and electron supply systems. Heterologous Ehrlich pathway genes, kivD encoding ketoisovalerate decarboxylase and adh encoding alcohol dehydrogenase, were constructed in S. oneidensis MR-1. Among the composition of carbon sources examined, 2% of N-acetylglucosamine, 1.5% of pyruvate and 2% of lactate were found to be the most optimal nutrients and resulted in 10.3 mg/L of isobutanol production with 48 h of microaerobic incubation. Finally, the effects of metal ions (electron acceptor) and direct electron transfer systems on isobutanol production were investigated, and Fe(2+) ions increased the isobutanol production up to 35%. Interestingly, deletion of mtrA and mtrB, genes responsible for membrane transport systems, did not have significant impact on isobutanol production. Finally, we applied engineered S. oneidensis to a bioelectrical reactor system to investigate the effect of a direct electron supply system on isobutanol production, and it resulted in an increased growth and isobutanol production (up to 19.3 mg/L). This report showed the feasibility of S. oneidensis MR-1 as a genetic host to produce valuable biochemicals and combine an electron-supplying system with biotechnological applications.

Temperature sensing using red fluorescent protein.

Genetically encoded fluorescent proteins are extensively utilized for labeling and imaging proteins, organelles, cell tissues, and whole organisms. In this study, we explored the feasibility of mRFP1 and its variants for measuring intracellular temperature. A linear relationship was observed between the temperature and fluorescence intensity of mRFP1 and its variants. Temperature sensitivities of E. coli expressing mRFP1, mRFP-P63A and mRFP-P63A[(4R)-FP] were -1.27%, -1.26% and -0.77%/°C, respectively. Finally, we demonstrated the potentiality of mRFP1 and its variants as an in vivo temperature sensor.

Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis.

The non-recyclable use of nitrogen fertilizers in microbial production of fuels and chemicals remains environmentally detrimental. Conversion of protein wastes into biofuels and ammonia by engineering nitrogen flux in Escherichia coli has been demonstrated as a method to reclaim reduced-nitrogen and curb its environmental deposition. However, protein biomass requires a proteolysis process before it can be taken up and converted by any microbe. Here, we metabolically engineered Bacillus subtilis to hydrolyze polypeptides through its secreted proteases and to convert amino acids into advanced biofuels and ammonia fertilizer. Redirection of B. subtilis metabolism for amino-acid conversion required inactivation of the branched-chain amino-acid (BCAA) global regulator CodY. Additionally, the lipoamide acyltransferase (bkdB) was deleted to prevent conversion of branched-chain 2-keto acids into their acyl-CoA derivatives. With these deletions and heterologous expression of a keto-acid decarboxylase and an alcohol dehydrogenase, the final strain produced biofuels and ammonia from an amino-acid media with 18.9% and 46.6% of the maximum theoretical yield. The process was also demonstrated on several waste proteins. The results demonstrate the feasibility of direct microbial conversion of polypeptides into sustainable products.