Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.

Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.

Chinese hamster ovary (CHO) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. Limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulting in enhanced growth and productivities in CHO cell fed-batch cultures (Mulukutla et al., 2017). In the current study, metabolic engineering of CHO cells was undertaken in order to reduce the biosynthesis of these novel growth inhibitors. Phenylalanine-tyrosine (Phe-Tyr) and branched chain amino acid (BCAA) catabolic pathways were engineered as part of this effort. Four genes that encode enzymes in the Phe-Tyr pathway, which were observed to be minimally expressed in CHO cells, were in turn overexpressed. Metabolically engineered cells were prototrophic to tyrosine and had reduced production of the inhibitory byproducts from Phe-Tyr pathway including 3-phenyllactate and 4-hydroxyphenyllactate. In case of BCAA catabolic pathway, branched chain aminotransferase 1 (BCAT1) gene, which encodes the enzyme that catalyzes the first step in the catabolism of BCAAs, was knocked out in CHO cells. Knockout (KO) of BCAT1 function completely eliminated production of inhibitory byproducts from BCAA catabolic pathway, including isovalerate, isobutyrate and 2-methylbutyrate, resulting in significantly enhanced cell growth and productivities in fed-batch cultures. This study is first of its kind to demonstrate that metabolic engineering of essential amino acid metabolism of CHO cells can significantly improve cell culture process performance.

A 3D-printed microbial cell culture platform with in situ PEGDA hydrogel barriers for differential substrate delivery.

Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200 µm. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.

A glimpse at the chemistry of GeH2 in solution. Direct detection of an intramolecular germylene-alkene π-complex.

The photochemistry of 3-methyl-4-phenyl-1-germacyclopent-3-ene (4) and a deuterium-labeled derivative (4-d(2)) has been studied in solution by steady state and laser flash photolysis methods, with the goal of detecting the parent germylene (GeH(2)) directly and studying its reactivity in solution. Photolysis of 4 in C(6)D(12) containing acetic acid (AcOH) or methanol (MeOH) affords 2-methyl-3-phenyl-1,3-butadiene (6) and the O-H insertion products ROGeH(3) (R = Me or Ac) in yields of ca. 60% and 15-30%, respectively, along with numerous minor products which the deuterium-labeling studies suggest are mainly derived from hydrogermylation processes involving GeH(2) and diene 6. The reaction with AcOH also affords H(2) in ca. 20% yield, while HD is obtained from 4-d(2) under similar conditions. Photolysis of 4 in THF-d(8) containing AcOH affords AcOGeH(3) and 6 exclusively, indicating that the nucleophilic solvent assists the extrusion of GeH(2) from 4 and alters the mechanism of the trapping reaction with AcOH compared to that in cyclohexane. Laser flash photolysis of 4 in hexanes yields a promptly formed transient exhibiting λ(max) ≈ 460 nm, which decays on the microsecond time scale with the concomitant growth of a second, much longer-lived transient exhibiting λ(max) ≈ 390 nm. The spectrum and reactivity of the 460 nm species toward various germylene trapping agents are inconsistent with those expected for free GeH(2); rather, the transient is assigned to an intramolecular Ge(II)-alkene π-complex of one of the isomeric substituted hydridogermylenes derived from a solvent-cage reaction between GeH(2) and its diene (6) coproduct, formed by addition of HGe-H across one of the C=C bonds. These conclusions are supported by the results of DFT calculations of the thermochemistry associated with π-complexation of GeH(2) with 6 and the formation of the isomeric vinylgermiranes and 1,2-hydrogermylation products. A different species is observed upon laser photolysis of 4 in THF solution and is assigned to the GeH(2)-THF complex on the basis of its UV-vis spectrum and rate constants for its reaction with AcOH and AcOD.

Direct detection of dimethylstannylene and tetramethyldistannene in solution and the gas phase by laser flash photolysis of 1,1-dimethylstannacyclopent-3-enes.

The photochemistry of 1,1-dimethyl- and 1,1,3,4-tetramethylstannacyclopent-3-ene (4a and 4b, respectively) has been studied in the gas phase and in hexane solution by steady-state and 193-nm laser flash photolysis methods. Photolysis of the two compounds results in the formation of 1,3-butadiene (from 4a) and 2,3-dimethyl-1,3-butadiene (from 4b) as the major products, suggesting that cycloreversion to yield dimethylstannylene (SnMe2) is the main photodecomposition pathway of these molecules. Indeed, the stannylene has been trapped as the Sn-H insertion product upon photolysis of 4a in hexane containing trimethylstannane. Flash photolysis of 4a in the gas phase affords a transient absorbing in the 450-520-nm range that is assigned to SnMe2 by comparison of its spectrum and reactivity to those previously reported from other precursors. Flash photolysis of 4b in hexane solution affords results consistent with the initial formation of SnMe2 (lambda(max) approximately 500 nm), which decays over approximately 10 micros to form tetramethyldistannene (5b; lambda(max) approximately 470 nm). The distannene decays over the next ca. 50 micros to form at least two other longer-lived species, which are assigned to higher SnMe2 oligomers. Time-dependent DFT calculations support the spectral assignments for SnMe2 and Sn2Me4, and calculations examining the variation in bond dissociation energy with substituent (H, Me, and Ph) in disilenes, digermenes, and distannenes rule out the possibility that dimerization of SnMe2 proceeds reversibly. Addition of methanol leads to reversible reaction with SnMe2 to form a transient absorbing at lambda(max) approximately 360 nm, which is assigned to the Lewis acid-base complex between SnMe2 and the alcohol.

Di- and trivalent organogermanium reactive intermediates. Kinetics and mechanisms of some reactions of diphenylgermylene and tetraphenyldigermene in solution.

The reactivity of diphenylgermylene (Ph2Ge) with several classes of germylene scavengers has been studied in hexane solution at 23 degrees C by laser flash photolysis of 3,4-dimethyl-1,1-diphenyl-1-germacyclopent-3-ene (1a), a clean and highly efficient precursor to the germylene and its dimer, tetraphenyldigermene (2a). The reactions studied include M-H insertion reactions with Group 14 hydrides (M = Si, Ge, Sn), halogen atom abstractions from bromo- and chlorocarbons, Lewis acid-base complexation with 1 degrees, 2 degrees, and 3 degrees aliphatic amines, and reaction with an aliphatic alkene, alkyne and diene, and oxygen. Absolute rate constants for (irreversible) scavenging of the germylene could be obtained by direct measurement of the germylene decay kinetics for all but the least efficient scavengers (triethylsilane, oxygen, chloroform, and 1-bromopentane), for which estimates of the rate constants were obtained by Stern-Volmer analysis of the reduction in digermene yield as a function of scavenger concentration. Distinctly different kinetic behavior is observed for scavenging of Ph2Ge by isoprene, 4,4-dimethyl-1-pentene, and triethylamine; in these cases, the results suggest that reaction is rapid (k(Q) = 3-6 x 10(9) M(-1)s(-1)) but reversible (K(eq) = 2500 - ca. 20,000 M(-1)) over the range of scavenger concentrations studied. The reactions with the C-C unsaturated compounds proceed via the intermediacy of long-lived transient species absorbing at <290 nm, which are tentatively assigned to the corresponding three-membered germanocycles on the basis of their UV spectra and lifetimes. Upper limits for the absolute rate constants for reaction of tetraphenyldigermene (2a) toward many of these reagents are also reported.

Organogermanium reactive intermediates. The direct detection and characterization of transient germylenes and digermenes in solution.

Diphenylgermylene (Ph2Ge) and its Ge=Ge doubly bonded dimer, tetraphenyldigermene (6a), have been characterized directly in solution for the first time by laser flash photolysis methods. The germylene is formed via (formal) cheletropic photocycloreversion of 3,4-dimethyl-1,1-diphenylgermacyclopent-3-ene (4a), which is shown to proceed in high chemical (>95%) and quantum yield (phi = 0.62) by steady-state trapping experiments with methanol, acetic acid, isoprene, and triethylsilane. Flash photolysis of 4a in dry deoxygenated hexane at 23 degrees C leads to the prompt formation of a transient assigned to Ph2Ge (lambda(max) = 500 nm; epsilon(max) = 1650 M(-1) cm(-1)), which decays with second-order kinetics (tau approximately 3 micros), with the concomitant growth of a second transient species that is assigned to digermene 6a (tau approximately 40 micros; lambda(max) = 440 nm). Analogous results are obtained from 1,1-dimesityl- and 1,1-dimethyl-3,4-dimethylgermacyclopent-3-ene (4b and 4c, respectively), which afford Mes2Ge (tau approximately 20 micros; lambda(max) = 560 nm) and Me2Ge (tau approximately 2 micros; lambda(max) = 480 nm), respectively, as well as the corresponding digermenes, tetramesityl- (6b; lambda(max) = 410 nm) and tetramethyldigermene (6c; lambda(max) = 370 nm). The results for the mesityl compound are compared to the analogous ones from laser flash photolysis of the known Mes2Ge/6b precursor, hexamesitylcyclotrigermane. The spectra of the three germylenes and two of the digermenes are in excellent agreement with calculated spectra, derived from time-dependent DFT calculations. Absolute rate constants for dimerization of Ph2Ge and Mes2Ge and for their reaction with n-butylamine and acetic acid in hexane at 23 degrees C are also reported.